TRBP control of PACT-induced phosphorylation of protein kinase R is reversed by stress.
The TAR RNA binding Protein, TRBP, inhibits the activity of the interferon-induced protein kinase R (PKR), whereas the PKR activator, PACT, activates its function. TRBP and PACT also bind to each other through their double-stranded RNA binding domains (dsRBDs) and their Medipal domains, which may influence their activity on PKR. In a human immunodeficiency virus (HIV) long terminal repeat-luciferase assay, PACT unexpectedly reversed PKR-mediated inhibition of gene expression. In a translation inhibition assay in HeLa cells, PACT lacking the 13 C-terminal amino acids (PACTDelta13), but not full-length PACT, activated PKR and enhanced interferon-mediated repression. In contrast, in the astrocytic U251MG cells that express low TRBP levels, both proteins activate PKR, but PACTDelta13 is stronger. Immunoprecipitation assays and yeast two-hybrid assays show that TRBP and PACTDelta13 interact very weakly due to a loss of binding in the Medipal domain. PACT-induced PKR phosphorylation was restored in Tarbp2(-/-) murine tail fibroblasts and in HEK293T or HeLa cells when TRBP expression was reduced by RNA interference. In HEK293T and HeLa cells, arsenite, peroxide, and serum starvation-mediated stresses dissociated the TRBP-PACT interaction and increased PACT-induced PKR activation, demonstrating the relevance of this control in a physiological context. Our results demonstrate that in cells, TRBP controls PACT activation of PKR, an activity that is reversed by stress.
Daher
Aïcha
A
Virus-Cell Interactions Laboratory, Lady Davis Institute for Medical Research, Montréal, Québec, Canada.
Laraki
Ghislaine
G
Singh
Madhurima
M
Melendez-Peña
Carlos E
CE
Bannwarth
Sylvie
S
Peters
Antoine H F M
AH
Meurs
Eliane F
EF
Braun
Robert E
RE
Patel
Rekha C
RC
Gatignol
Anne
A
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D001253:Astrocytes / Q000187:drug effects / Q000201:enzymology; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D016325:HIV Long Terminal Repeat / Q000235:genetics; D006801:Humans; D007372:Interferons / Q000494:pharmacology; D051379:Mice; D010766:Phosphorylation / Q000187:drug effects; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000187:drug effects; D034622:RNA Interference / Q000187:drug effects; D016601:RNA-Binding Proteins / Q000378:metabolism*; D017384:Sequence Deletion; D013312:Stress, Physiological / Q000187:drug effects; D019892:eIF-2 Kinase / Q000378:metabolism*; D000099253:Trans-Activation Responsive RNA-Binding Protein
D000952:Antigens, Polyomavirus Transforming; D007372:Interferons; D016601:RNA-Binding Proteins; D019892:eIF-2 Kinase; D000099253:Trans-Activation Responsive RNA-Binding Protein; C104647:PRKRA protein, human; C119610:Prkra protein, mouse